Extended confocal microscopy of myocardial laminae and collagen network

Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a techniq ue for imaging the 3D organization of myocytes in conjunction with the coll agen network in extended blocks of myocardium. Rat hearts were fixed with B ouin's solution and perfusion-stained with picrosirius red. Transmural bloc ks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser sc anning microscopy was used to obtain 3D images to a depth of 60 mu m in a c ontiguous mosaic across the surface, Approximately 50 mu m was then cut off the surface of the block with an ultramicrotome. This sequence was repeate d 20 times. Images were assembled and registered in 3D to form an extended volume 3800x800x800 mu m(3) spanning the heart wall from epicardium to endo cardium. Examples are given of how digital reslicing and volume rendering m ethods can be applied to the resulting dataset to provide quantitative stru ctural information about the 3D organization of myocytes, extracellular col lagen matrix and blood vessel network of the heart.